Meat tenderization

ABSTRACT

The present invention relates to methods for tenderizing meat comprising contacting meat with a tenderizing-effective amount of a thermolabile protease having limited substrate specificity, wherein the limited substrate specificity is the digestion of only one of the two major protein components of meat.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/773,534, filed Feb. 5, 2004, which is a continuation of U.S.application Ser. No. 10/041,080, filed Apr. 18, 2002, which is acontinuation of U.S. application Ser. No. 09/620,494, filed Jul. 20,2000, which is a continuation of U.S. application Ser. No. 09/358,792(now U.S. Pat. No. 6,149,950), filed on Jul. 22, 1999, the contents ofwhich are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for meattenderization employing thermolabile enzymes having limited substratespecificity.

BACKGROUND OF THE INVENTION

Tenderness is a major quality attribute that affects market price andconsumer acceptance of meat products. The lack of consistency of beeftenderness is a major reason for decreased beef consumption in the U.S.There are several methods for improving meat tenderness, includingmechanical tenderization, elevated temperature storage, calcium chlorideinjection, electrical stimulation, muscle stretching, shock-wavepressure, dry and wet aging, and enzymatic treatment. One of the mostwidely used is treatment with an enzyme, such as, e.g., papain,bromelain, or ficin. These enzymes, however, have very broadspecificities and therefore hydrolyze indiscriminately the major meatproteins (connective tissue/collagen and myofibrillar proteins)resulting in an over-tenderized (i.e., mushy) product. Furthermore,papain, which is the most widely used, is relatively heat-stable,allowing uncontrolled texture deterioration during and after cooking.

Thus, there is a need in the art for meat tenderization methodsemploying enzymes which, unlike papain, have narrow substratespecificity; express self-limiting hydrolysis of meat proteins;hydrolyze either of the two major meat protein components, but not both;are thermolabile and thereby readily inactivated at cookingtemperatures; and have no adverse effect on flavor.

SUMMARY OF THE INVENTION

The present invention provides methods for tenderizing meat, which arecarried out by contacting meat with a solution comprising atenderizing-effective amount of a thermolabile protease. Preferably, theprotease has a limited substrate specificity so that it (a) digests meatproteins to only a limited extent and/or (b) digests only one of the twomajor protein components of meat. In some embodiments, the protease isderived from a Rhizomucor species, preferably R. miehei. In someembodiments, the protease is derived from a mammal, such as, e.g.,bovine chymosin. The protease may be isolated directly from R. mieheicells or, alternatively, from recombinant host cells transformed withnucleic acid encoding the protease. Preferably, the protease is treatedwith peroxy acids to render it more thermolabile.

The contacting step comprises, without limitation, injection of meat;marination; or injection of an animal prior to slaughter, and may alsoinclude tumbling the contacted meat in the tenderizing solution.Typically, meat is contacted with the protease at a weight ratio ofbetween about 0.001 and about 0.1 AU/100 g meat, preferably betweenabout 0.005 and about 0.05 AU/100 g meat.

The methods of the invention provide tenderized meat that exhibits arelative shear force (as measured by the Warner-Bratzler method) that isbetween about 50% and about 90%, preferably between about 60% and about80%, of the relative shear force of the meat prior to tenderization.

In another aspect, the invention provides tenderized meat productsproduced using the methods described herein.

In yet another aspect, the invention provides meat tenderizingcompositions comprising a thermolabile protease having limited substratespecificity and one or more of brine, curing agents, and flavoringagents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the thermolability ofthermolabile R. miehei protease (diamond symbols) and papain (boxsymbols).

FIG. 2 is a schematic representation of the relative shear force of meattreated with thermolabile R. miehei protease (diamond symbols) andpapain (circle symbols).

FIG. 3 is a schematic representation of the effect of refrigeratedstorage on the relative shear force of tenderized meat. Diamond symbols,control; box symbols, thermolabile R. miehei protease; triangle symbols,papain.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for tenderizingmeat. The invention is based on the discovery that treating meat withthermolabile proteases having limited substrate specificity results inmeat that exhibits superior properties relative to untenderized meat orto meat treated with conventional tenderizing enzymes such as, e.g.,papain.

Proteases:

As used herein, a thermolabile protease refers to a protease whoseenzymatic activity is expressed at reduced levels at high temperatures,such as, e.g., above about 50° C., due to (a) thermolability of theenzymatic reaction catalyzed by the protease and/or (b) structuralthermolability of the protease itself. According to the invention, athermolabile protease exhibits less than about 75%, preferably less thanabout 50%, more preferably less than about 25%, and most preferably lessthan about 10%, of its maximal enzymatic activity when the enzymaticreaction is carried out at a temperature of at least about 70° C.,preferably at least about 60° C. and more preferably at least about 50°C. Alternatively, or in combination with the above, a thermolabileprotease is a protease whose activity declines to less than about 75%,preferably less than about 50%, more preferably less than about 25%, andmost preferably less than about 10%, of its maximal enzymatic activityafter incubation for 5-30 min at a temperature of at least about 70° C.,preferably at least about 60° C., and more preferably at least about 50°C., when enzymatic activity is measured at the temperature at which theuntreated enzyme normally exhibits its maximal activity.

As used herein, a protease having limited substrate specificity is onethat recognizes only a subset of amino acids within a particularpolypeptide as targets for proteolytic attack. Proteases having limitedsubstrate specificity that are useful in practicing the presentinvention include, without limitation, those that (a) digest either orboth of the meat protein components to a degree of hydrolysis (DH) ofless than about 10%, preferably less than about 5%, and most preferablyless than about 2%, and/or (b) digest only one of the two major proteincomponents of meat, when meat is contacted as described, e.g., inExample 2 below. DH may be measured using any method known in the art,including, without limitation, measuring free amino groups using the OPA(o-phthaldialdehyde) method (Church et al., Anal. Biochem. 146:343,1985) (see, Example 3 below); measuring a decrease in pH; and measuringan increase in osmolality. Methods for comparing hydrolysis of collagenand myofibrillar proteins are described, e.g., in Example 4 below.

Proteases for use in the present invention may comprise wild-type ormutant enzymes. The enzymes may be isolated from their cell of origin ormay be recombinantly produced using conventional methods well-known inthe art. The only requirement is that the protease exhibitthermolability and, preferably, limited substrate specificity and becapable of exerting a tenderizing effect on meat as described herein.

In one embodiment, the protease used in practicing the present inventionis derived from a Rhizomucor species, including, without limitation,Rhizomucor miehei or Rhizomucor pusillus, preferably R. miehei. Thesequence of R. miehei protease is disclosed in U.S. Pat. No. 5,800,849.

In another embodiment, the protease used in practicing the presentinvention is derived from a mammal, such as, e.g., bovine chymosin(which is commercially available from, e.g., Chr. Hansen, A/S, Denmark).

The enzyme may be inherently thermolabile or may be renderedthermolabile by any means known in the art, including, withoutlimitation, chemical or other treatments of the native enzyme, or byintroduction of mutations into the sequence of the enzyme that result inenhanced thermolability. In some embodiments, R. miehei protease isrendered more thermolabile by treatment with oxidizing agents containingactive chlorine (U.S. Pat. No. 4,357,357) or aliphatic or inorganicperoxy acids (U.S. Pat. No. 4,591,565).

Methods for Tenderizing Meat:

The present invention provides methods for tenderizing meat thatcomprise contacting meat with a solution comprising atenderizing-effective amount of a thermolabile protease. In preferredembodiments, the protease has a limited substrate specificity.

As used herein, “meat” encompasses, without limitation, meat muscle,whether present in a live animal or carcass or in fresh meat obtainedfrom butchering, as well as frozen meat, freeze-dried meat, andrestructured meat in any form.

Tenderizing refers to a process by which the texture of meat is renderedmore chewable or otherwise more acceptible to the consumer.Tenderization is typically assessed by measuring the relative shearforce of the meat or by sensory evaluation. Relative shear force may bemeasured using the Warner-Bratzler method (Olson et al., J. Food Sci.42:506, 1977; and Fogle et al., J. Food Sci. 47:1113, 1982). In thismethod, meat samples are carved into cylindrical shapes 0.75 cm indiameter with the axis of the cylinder along the longitudinal sectionsof the meat fibers. The samples are placed on a V-shaped Warner-Bratzlerblade moving down at a steady speed of 2 mm/sec through a fixed distance(30-50 mm) slicing along the length of the sample. The maximum forcerequired to slice through each sample is an indication of the shearforce.

A tenderizing-effective amount of a protease is an amount that resultsin a reduction of relative shear force to between about 50-90%,preferably between about 60-80%, of the relative shear force exhibitedby untreated meat.

In practicing the invention, meat is contacted with the enzyme at aratio (by weight) of between about 0.001 and about 0.1 Anson Units(AU)/100 g meat, preferably between about 0.0025 and about 0.05 AU/100 gmeat and most preferably between about 0.005 and about 0.05 AU/100 gmeat. One AU is defined as the amount of enzyme which digests denaturedhemoglobin at 25° C., pH 7.5 in 10 min, at an initial rate thatliberates an amount of trichloroacetic acid-soluble material that isequivalent to one milliequivalent of tyrosine, when measured by colorproduction using a phenol reagent.

The contacting step may comprise one or more of (a) injecting the meatdirectly; (b) marinating the meat; or (c) injecting a live animal priorto slaughter. It will be understood that the time during which the meatis contacted with the protease, as well as the temperature at which themeat is contacted, will depend upon the mode (or combination of modes)that is employed. For example, meat may be injected with aprotease-containing solution and stored for up to about two weeks at 5°C. Alternatively, meat may be marinated for about 0.5-10 h at 5-10° C.Furthermore, the meat will be contacted with the protease duringcooking.

The method may further comprise tumbling the meat in, e.g., themarinating solution, such as, e.g., for 1-3 h at 5-10° C.

It will be understood that each of the reaction conditions (such as,e.g., concentration of protease, ratio of protease:meat, mode ofcontacting, pH, temperature, and time) may be varied, depending upon thesource of meat and/or enzyme and the degree of tenderization that isrequired. It will further be understood that optimization of thereaction conditions may be achieved using routine experimentation byestablishing a matrix of conditions and testing different points in thematrix.

In other embodiments, the invention encompasses meat and meat productsthat have been tenderized using the above-described methods andcompositions.

In other embodiments, the invention encompasses compositions for meattenderization that comprise, in addition to a thermostable protease, oneor more of brine, curing agents, and flavoring agents. Brine comprises,without limitation, sodium chloride, phosphates, dextrose, and otheringredients. Curing agents include, without limitation, nitrites,sugars, and erythorbate. Flavoring agents include, without limitation,herbs, spices, and liquid smoke.

The following examples are intended as non-limiting illustrations of thepresent invention.

EXAMPLE 1 Thermolability of R. miehei Protease

The following experiment was performed to compare the thermolability ofR. miehei protease with that of papain, the most widely used enzyme formeat tenderization.

Enzymes were heat-treated for 30 min at 50, 60, and 70° C., after whichtheir residual activity was determined as follows, using a Protazyme kit(Megazyme International Ireland Ltd.)

1-ml enzyme (in 100 mM disodium hydrogen phosphate, 30 mM cysteinhydrochloride, 30 mM EDTA, pH 7.0) were added to 1 mlprotazyme-containing buffer (100 mM disodium hydrogen phosphate, 1% w/vsodium dodecyl sulfate, pH 7.0) and incubated for 10 min at 40° C. withcontinuous stirring, after which the reaction was terminated by additionof 10 ml 2%(w/v) trisodium phosphate pH 12.3 with vigorous stirring. Thereactions were allowed to stand at room temperature for a further 2 min,after which they were filtered through a Whatman No. 2 filter. Theabsorbance of the supernatant at 590 nm was measured against a substrateblank (which contained the same components, except that 1 ml of bufferwas added instead of enzyme).

The results (FIG. 1) indicate that thermolabile-R. miehei proteaserapidly loses activity at temperatures greater than 60° C.

EXAMPLE 2 Meat Tenderization Using R. miehei Protease

The following experiment was performed to evaluate meat tenderizationusing R. miehei protease.

Methods:

Beef (top round post rigor meat, i.e., 72 hr postmortem) was purchasedfrom a local abattoir and used fresh. Samples were weighed and injectedwith enzyme solution to provide a net gain of 5% (wet meat weight) at apressure of 50 psi with injection needles being about 1 inch apart. Themeat was tumbled for 2 hr at 7 rpm in a walk-in cooler (5° C.) afterwhich samples were sliced into portions of 1″ thickness.

Samples were stored for 1 week at 5° C. and analyzed on days 1, 7, and14, after which Warner-Bratzler shear force was measured as follows:Core samples (1″ diameter) from cooked meat were subjected toWarner-Bratzler shear force in a TA-XT2 Texture Analyzer and the maximumforce estimated as indicator of tenderness. Test speed was 2 mm/sthrough a fixed distance of 30 mm.

Results:

Of the two enzymes tested, papain exhibited the greater effect on beeftenderness. At the lowest concentration, papain reduced tenderness bymore than 40% and reduced the meat to mush at higher concentrations,making it impossible to obtain any tenderness evaluations (FIG. 2). Bycontrast, thermolabile R. miehei protease provided an optimal level ofmeat tenderization. Furthermore, use of this enzyme allows refrigeratedstorage of tenderized meat without further hydrolytic degradation of themeat (FIG. 3).

EXAMPLE 3 Measurement of Degree of Hydrolysis (DH)

Protein hydrolysates are analyzed for DH by OPA as follows: The OPAreagent is prepared by dissolving 7.620 g di-sodium tetraboratedecahydrate (Aldrich 22, 133-3) and 200 mg sodium dodecyl sulphate(Sigma L-3771) in 150 ml water. 160 mg o-phthaldialdehyde 97% (SigmaP-0657) is dissolved in 4 ml ethanol and added to the mixture, broughtto 200 ml with deionized water. 3 ml OPA reagent is added to a testtube, after which 400 μl serine standard or sample is added. Aftermixing, the mixtures are incubated for exactly 2 minutes, after whichabsorbance at 340 nm is measured. DH is calculated using the followingformulas:$\quad{{{a.{Serine}}\quad{NH}_{2}} = {\frac{{OD}_{sample} - {OD}_{blank}}{{OD}_{standard} - {OD}_{blank}}*0.9516\quad{meq}\quad v\text{/}l*\frac{0.1*100\quad l\text{/}g\quad{protein}}{X*P}}}$

-   -   Serine NH₂=meqv serine NH₂/g protein    -   X=g sample    -   P=% protein in sample    -   0.1=sample volume in liters        $h = {\frac{{Serine} - {NH}_{2} - ß}{\alpha}\quad{meq}\quad v\text{/}g\quad{protein}}$    -   b. DH=h/h_(tot)*100%

EXAMPLE 4 Measurement of Hydrolytic Potential of a Protease for Collagenand Myofibrils

The following experiments are performed to evaluate the relativeactivity of a protease on each of the two major protein components ofmeat.

Collagenase Activity:

-   1. To 20 mg collagen from bovine tendon (Sigma) suspended in 3.8 ml    Tris buffer (0.02 M Tris, 0.005 M CaCl₂, pH 7.4) is added 200 μl    collagenase (or protease) solution (1 mg/ml in Tris buffer) to make    a total volume of 4.0 ml.-   2. The mixture is incubated at 40° C. for 3 hr or 70° C. for 30 min.-   3. The reaction mixtures are centrifuged in a microfuge for 10 min    at 14,000 rpm.-   4. 1.5 ml of supernatant is mixed with 4.5 ml of 5 N HCl and kept in    a drying oven at 110° C. for 16 hrs (overnight) for complete    hydrolysis of soluble peptides.-   5. The hydrolysate is then analyzed for hydroxyproline content as    follows:    Hydroxyproline Content-   1. The hydrolysate is diluted 25 times with distilled water.-   2. To 1.00 ml of diluted hydrolysate 1.00 ml of chloramine-T    solution is added and the mixture is allowed to stand at room    temperature for 20 min.-   3. 1.00 ml of color reagent are added after this period and the    reaction mixture is transferred to a 60° C. water bath and incubated    for 15 min.-   4. Tubes are removed and allowed to cool down to room temperature.-   5. Absorbance at 560 nm is measured.    Myofibril Extraction (Olson et al., 1976, J. Food Sci. 41:    1036-1041).-   1. 100 g of minced meat is homogenized for 30 sec in 10 vols (v/w)    KCl-phosphate buffer (100 mM KCl, 20 mM potassium phosphate—pH 7.0)    at 11,000 rpm.-   2. The homogenate is centrifuged at 1,000 g for 15 min and the    pellet re-suspended in 5 vols (v/w) buffer and re-centrifuged under    the same conditions.-   3. The pellet is again re-suspended in 5 vols buffer and poured    through strainer to remove connective tissue and debris.-   4. The myofibril suspension obtained is centrifuged at 1,000 g for    15 min, re-suspended, and washed twice more with buffer.-   5. The myofibril extract is suspended in buffer and protein content    estimated using the Leco protein analyzer.    Soluble Protein Analysis-   1. To 2.9-3.0 ml myofibril extract 0-100 μl enzyme solution (1 mg    protein/ml) are added, and the reaction mixtures incubated at 70° C.    for 30 min.-   2. The reaction is stopped by adding 1.5 ml of 15% trichloroacetic    acid and allowing the mixtures to stand at room temperature for 15    min.-   3. The suspension is centrifuged at 10,000 rpm for 8 min and the    absorbance of the superatanat at 280 nm is measured.

Alternatively,

-   1. 50 μl enzyme (1 mg protein/ml) is added to 1.95 ml of myofibril    extract and incubated at 70° C. for 30 min.-   2. The reaction is stopped by adding 2 ml of 15% trichloroacetic    acid and allowing the mixture to stand at room temperature for 15    min.-   3. The suspension is centrifuged at 10,000 rpm for 8 min and the    absorbance of supernatant at 280 nm is measured.

EXAMPLE 5 Thermolability of Bovine Chymosin

The following experiment was performed to test the thermolability ofbovine chymosin.

The enzyme (Chy-Max from Chris Hansen, Inc.) was heat-treated for 30 minat 55, 65, and 75° C., after which its residual activity (expressed inAnson Units using hemoglobin as a substrate) was determined using aNeutrase standard at pH 7.5.

The results indicate that bovine chymosin rapidly loses activity attemperatures greater than 60° C. and retains only 10-15% activity afterheating to 75° C.

All patents, patent applications, and literature references referred toherein are hereby incorporated by reference in their entirety.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed description.Such obvious variations are within the full intended scope of theappended claims.

1-20. (canceled)
 21. A method for tenderizing meat, said methodcomprising contacting meat with a tenderizing-effective amount of aprotease having limited substrate specificity, wherein said limitedsubstrate specificity is the digestion of only one of the two majorprotein components of meat.
 22. A method as defined in claim 21, whereinsaid protease has a limited substrate specificity.
 23. A method asdefined in claim 21, wherein said protease is derived from a Rhizomucorspecies.
 24. A method as defined in claim 23, wherein said Rhizomucorspecies is R. miehei.
 25. A method as defined in claim 24, wherein saidprotease is treated with peroxy acids prior to said contacting.
 26. Amethod as defined in claim 21, wherein said protease is derived from amammal.
 27. A method as defined in claim 26, wherein said mammal isbovine.
 28. A method as defined in claim 27, wherein said protease ischymosin.
 29. A method as defined in claim 21, wherein said protease isobtained from a recombinant host cell transformed with a nucleic acidencoding said protease.
 30. A method as defined in claim 21, whereinsaid meat after tenderization exhibits a relative shear force of betweenabout 50% and about 90% of said meat prior to tenderization.
 31. Amethod as defined in claim 30, wherein said meat after tenderizationexhibits a relative shear force of between about 60% and about 80% ofsaid meat prior to tenderization.
 32. A method as defined in claim 21,wherein said contacting comprises injection or marination.
 33. A methodas defined in claim 32, further comprising tumbling said meat.
 34. Amethod as defined in claim 21, wherein said meat is contacted with saidprotease at a ratio of between about 0.001 and about 0.05 AU/g meat. 35.A method as defined in claim 21, wherein said meat is selected from thegroup consisting of fresh meat, frozen meat, freeze-dried meat, andrestructured meat.